The present invention also relates to the use of an enzyme, in particular xcex1-1,4-glucan lyase (xe2x80x9cGLxe2x80x9d), to prepare 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d) from substrates based on xcex1-1,4-glucan.
The present invention also relates to the use of a sugar, in particular 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d), as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.
The present invention relates to the use of 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d) as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.
FR-A-2617502 and Baute et al in Phytochemistry [1988] vol. 27 No. 11 pp3401-3403 report on the production of AF in Morchella vulgaris by an apparent enzymatic reaction. The yield of production of AF is quite low. Despite a reference to a possible enzymatic reaction, neither of these two documents presents any amino acid sequence data for any enzyme let alone any nucleotide sequence information. These documents say that AF can be a precursor for the preparation of the antibiotic pyrone microthecin.
Yu et al in Biochimica et Biophysica Acta [1993] vol 1156 pp313-320 report on the preparation of GL from red seaweed and its use to degrade xcex1-1,4-glucan to produce AF. The yield of production of AF is quite low. Despite a reference to the enzyme GL this document does not present any amino acid sequence data for that enzyme let alone any nucleotide sequence information coding for the same. This document also suggests that the source of GL is just algal.
A typical xcex1-1,4-glucan based substrate is starch. Today, starches have found wide uses in industry mainly because they are cheap raw materials.
Starch degrading enzymes can be grouped into various categories. The starch hydrolases produce glucose or glucose-oligomers. A second group of starch degrading enzymes are phosphorylases that produce glucose-1-phosphate from starch in the presence of inorganic phosphate.
AF has also been chemically synthesisedxe2x80x94see the work of Lichtenthaler in Tetrahedron Letters Vol 21 pp 1429-1432. However, this chemical synthesis involves a large number of steps and does not yield large quantities of AF.
The chemical synthetic route for producing AF is therefore very expensive.
There is therefore a need for a process that can prepare AF in a cheap and easy manner and also in a way that enables large quantities of AF to be made.
Furthermore, anti-oxidants are typically used to prevent oxygen having any deleterious effect on a substance such as a foodstuff. Two commonly used anti-oxidants are GRINDOX 142 and GRINDOX 1029. These anti-oxidants contain many components and are quite expensive to make.
There is therefore a need to have a simpler and cheaper form of anti-oxidant.
Furthermore, sweeteners are often used in the preparation of foodstuffs and beverages. However, many sweeteners are expensive and complex to prepare.
There is therefore a need to have a simpler and cheaper form of sweetener.
According to the present invention there is provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an xcex1-1,4-glucan with the enzyme xcex1-1,4-glucan lyase characterised in that enzyme is used in substantially pure form.
Preferably if the glucan contains links other than and in addition to the xcex1-1,4-links the xcex1-1,4-glucan lyase is used in conjunction with a suitable reagent that can break the other linksxe2x80x94such as a hydrolasexe2x80x94preferably glucanohydrolase.
Preferably the glucan is starch or a starch fraction prepared chemically or enzymatically. If prepared enzymatically the reaction can be performed before the addition of the xcex1-1,4-glucan lyase or the reactions can be performed simultaneously. The suitable reagent can be an auxiliary enzyme. Preferred auxiliary enzymes are alpha- or beta-amylases. Preferably a debranching enzyme is used. More preferably the auxiliary enzyme is at least one of pullanase or isoamylase.
Preferably the xcex1-1,4-glucan lyase either is bound to a support or, more preferably, is in a dissolved form.
Preferably the enzyme is isolated from either a fungus, preferably Morchella costata or Morchella vulgaris, or from a fungally infected algae, preferably Gracilariopsis lemaneiformis, or from algae lone, preferably Gracilariopsis lemaneiformis. 
Preferably the enzyme is isolated and/or further purified from the fungus or from the fungally infected algae or algae alone using a gel that is not degraded by the enzyme.
Preferably the gel is based on dextrin or derivatives thereof.
Preferably the gel is a cyclodextrinxe2x80x94more preferably beta-cyclodextrin. Preferably the enzyme comprises the amino acid sequence SEQ. I.D. No. 1. or the amino acid sequence SEQ. I.D. No. 2 or the amino acid sequence SEQ. ID. No. 5 or the amino acid SEQ. I.D. No. 6, or any variant thereof.
In an alternative preferable embodiment, the enzyme comprises any one of the amino acid sequences shown in SEQ. I.D. No.s 9-11, or any variant thereof.
The term xe2x80x9cany variant thereofxe2x80x9d means any substitution of, variation of, modification of, replacement of, deletion of or addition of an amino acid from or to the sequence providing the resultant enzyme has lyase activity.
Preferably the enzyme is used in combination with amylopectin or dextrin.
Preferably, the enzyme is obtained from the expression of a nucleotide sequence coding for the enzyme.
Preferably the nucleotide sequence is a DNA sequence.
Preferably the DNA sequence comprises a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8.
In an alternative preferable embodiment, the DNA sequence comprises any one of the sequences that are the same as, or are complementary to, or have substantial homology with, or contain any suitable codon substitutions as shown as SEQ. ID. No.s 12-14.
The expression xe2x80x9csubstantial homologyxe2x80x9d covers homology with respect to structure and/or nucleotide components and/or biological activity.
The expression xe2x80x9ccontains any suitable codon substitutionsxe2x80x9d covers any codon replacement or substitution with another codon coding for the same amino acid or any addition or removal thereof providing the resultant enzyme has lyase activity.
In other words, the present invention also covers a modified DNA sequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode a polypeptide having the activity of a glucan lyase, preferably having an increased lyase activity.
Preferably the starch is used in high concentrationxe2x80x94such as up to about 25% solution.
Preferably the substrate is treated with the enzyme in the presence of a buffer.
More preferably the substrate is treated with the enzyme in the presence of substantially pure water.
Preferably the substrate is treated with the enzyme in the absence of a co-factor.
According to the present invention there is also provided a method of preparing the sugar 1,5-D-anhydrofructose comprising treating an xcex1-1,4-glucan with the enzyme xcex1-1,4-glucan lyase characterised in that enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino acid sequence SEQ. ID. No. 6, or any one of the amino acid sequences SEQ. I.D. No.s 9-11, or any variant thereof.
According to the present invention there is also provided the sugar 1,5-D-anhydrofructose when prepared by the method of the present invention.
AF prepared by the present method was confirmed and characterised by 13C NMR.
One of key advantages of the present method is that the sugar 1,5-D-anhydrofructose can be prepared in much larger quantities than before and by a method that is relatively easier and cheaper than the known processes. For example the sugar can now be prepared in amounts of for example greater than 100 gxe2x80x94such as 500 gxe2x80x94compared to the prior art methods when only much smaller amounts were and could be producedxe2x80x94such as micro gram amounts.
Typical reactions that can be catalyzed by GL can be summarised as follows:
1). Amylopectinxe2x86x92AF+limit dextrin
2). Amylosexe2x86x92AF+limit dextrin
3). Dextrinxe2x86x92AF+glucose
In reaction 1), the ratio of the two products depend on the structure of amylopectin or the distribution of xcex1-1,6glucosidic linkages in the amylopectin molecules.
In reaction 2) and 3), the ratio of the products depends on the degree of polymerisation (DP) number of the substrate. In reaction 3 the ratio between AF and glucose depends upon the DP. For example if the dextrin contains 10 glucose units the ratio AF:glucose would be 9:1.
Another advantage of the present invention is that glucans that contain links other than xcex11,4-links can be substantially degradedxe2x80x94whereas before only partial degradation was achieved. The substantial degradation of the 1,5-D-anhydrofructose precursor is one of the factors leading to the increased yields of 1,5-D-anhydrofructose.
Other advantages are AF is a naturally occurring substance and therefore it has a potential for human purposes. For example, it can be converted to the antibiotic microthecin by AF dehydrase. Antibiotics are known for their uses in food bio-preservation, which is an important area in food technology. However, to date, the preparation of AF and also microthecin has had a number of disadvantages. For example, only small quantities could be produced. Also, the process was costly.
The present invention overcomes these problems by providing a larger production of and much cheaper production of AF and so also other products such as microthecin. In this regard, it is possible to prepare gram to kilogram amounts of AF.
A further advantage is that the lyase is stable for at least one year at 4xc2x0 C. and can be lyophilized without loss of activity.
Another advantage is that the lyase produces AF directly from starches and does not need the presence of any co-factors.
Another advantage is that the enzyme can be used in pure water. This result is very surprising.
Based on the simple properties of the present lyase, one can expect that the production cost of AF will be comparable to that of glucose. This is especially advantageous that the present lyase does not necessarily require the presence of any co-factors which are generally very expensive.
In general xcex1-1,4-glucans can be used as substrate for the enzyme.
As a preferred substrate, starch is used.
In a preferred process, soluble or gelatinized starch or starch hydrolysate are used. The starch hydrolysates can be prepared either chemically or enzymatically.
If an enzyme is used for the partial starch degradation the enzyme can either be added before the addition of the lyase or any other additional starch degrading reagent (such as the enzyme glucanohydrolase) which may be added simultaneously.
The lyase will convert the glucan to AF. The enzyme will attach the substrate from the non reducing end and leave only the reducing sugar unconverted. The residual glucose can be removed by known methods some of which have been described here.
Using the reaction described here pure AF can be produced and also in large amounts.
In one embodiment, the xcex1-1,4-glucan lyase is purified from the fungally infected algaexe2x80x94such as Gracilariopsis lemaneiformisxe2x80x94by affinity chromatography on xcex2-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from xcex1-1,4-glucans.
The fungal lyase isolated from fungal infected Gracilariopsis lemaneiformis is characterized as having a pH optimum at 3.5-7.5 when amylopectin is used, a temperature optimum at 50xc2x0 C. and a pI of 3.9.
In another embodiment, the xcex1-1,4-glucan lyase is purified from the fungus Morchella costata by affinity chromatography on xcex2-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 55 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from xcex1-1,4-glucans.
The fungal lyase shows a pI around 5.4 as determined by isoelectric focusing on gels with pH gradient of 3 to 9. The molecular weight determined by SDS-PAGE on 8-25% gradient gels was 110 kDa. The enzyme exhibited a pH optimum in the range pH 5-7. The temperature optimum was found to be between 30-45xc2x0 C.
In another embodiment, the xcex1-1,4-glucan lyase is purified from the fungus Morchella vulgaris by affinity chromatography on xcex2-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from xcex1-1,4-glucans.
In another embodiment, the xcex1-1,4-glucan lyase is purified from algaexe2x80x94such as Gracilariopsis lemaneiformisxe2x80x94by affinity chromatography on xcex2-cyclodextrin Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel filtration on Superose 12 columns. The purified enzyme produces 1,5-anhydro-D-fructose from xcex1-1,4-glucans.
Typical pH and temperature optima for the lyase catalyzed reaction for some of the GL enzymes according to the present invention are as follows:
The enzymes of the present invention convert amylose and amylopectin to 1,5-anhydrofructose.
Among the maltosaccharides tested, we found that the lyase showed low activity towards maltose, and lower activity to maltotriose and maltoheptaose with the highest activity to maltotetraose and maltopentaose. The enzyme showed no substrate inhibition up to a concentration 10 mg mlxe2x88x921 among these maltosaccharides.
The enzymes from each of the preferred sources has been sequenced and the amino acid sequences are presented later. Also presented later are the DNA sequences coding for the enzymes.
The present invention therefore describes a new starch degrading enzymexe2x80x94namely a new xcex1-1,4-glucan lyase. This is an enzyme that has been purified and characterized for the first time.
As mentioned above, the present invention also relates to some specific uses of AF.
In particular, the present invention relates to the use of 1,5-D-anhydrofructose (xe2x80x9cAFxe2x80x9d), as an anti-oxidant, in particular as an anti-oxidant for food stuffs and beverages.
Therefore according to the present invention there is provided the use of 1,5-D-anhydrofructose (AF) as an anti-oxidant.
Preferably AF is or is used in an edible substance.
Preferably AP is used in or as a foodstuff or beverage.
Preferably, AF is used in combination with another anti-oxidant.
Preferably the AF is prepared by the method according to the present invention.
The main advantages of using AF as an anti-oxidant are that it is a natural product, it is non-metabolisable, it is easy to manufacture, it is water-soluble, and it is generally non-toxic.
In a preferred embodiment the present invention therefore relates to the enzymatic preparation of pure AF which can be used as an attractive water soluble antioxidant for food and non-food purposes. In the application examples are given for the use of AF as an antioxidant in food formulations.
In the accompanying examples it is seen that AF is comparable with known high quality commercial available food antioxidants. Non-food examples include use in polymer chemistry as oxygen scavengers during the synthesis of polymers. Also, AF could be used for the synthesis of bio-degradable plastic.
Experiments have shown that AF can be an efficient reducing agent (antioxidant), as it can easily reduce 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid.
AF is a naturally occurring substance and therefore it has a tremendous potential for use as an acceptable antioxidant. AF can also be converted into the antibiotic microthecin by AF dehydrase. Antibiotics are known for their uses in food biopreservation, an important area in food biotechnology.
In another aspect, the present invention also relates to the use of 1,5-D-anhydrofructose as a sweetener, in particular as a sweetener for foodstuffs and beverages, preferably human foodstuffs and beverages.
Thus according to this aspect of the present invention there is provided the use of 1,5-D-anhydrofructose as a sweetener.
Preferably the AF is used as or in a human foodstuff or beverage.
The AF may be used in any desired amount such as a 5% solution or 100 mg/kg to 500 mg/kg.
The advantages of using AF as a sweetener are that it is a natural product, it is generally non-toxic, it is water soluble, it is non-metabolisable and it is easy to manufacture.
The present invention therefore also relates to a novel application of AF as a sweetener.
Preferably the AF is prepared by the method according to the present invention.
Further aspects of the present invention include:
a method of preparing the enzyme xcex1-1,4-glucan lyase (GL) comprising isolating the enzyme from a fungally infected algae, fungus or algae alone;
an enzyme comprising the amino acid sequence SEQ. ID. No. 1. or SEQ. ID. No. 2 or SEQ. ID. No. 5. or SEQ. ID. No. 6, or any variant thereof;
an enzyme comprising the amino acid sequence SEQ. ID. No. 9. or SEQ. ID. No. 10 or SEQ. ID. No. 11, or any variant thereof;
a nucleotide sequence coding for the enzyme xcex1-1,4-glucan lyase, preferably wherein the sequence is not in its natural environment (i.e. it does not form part of the natural genome of a cellular organism capable of expressing the enzyme, preferably wherein the nucleotide sequence is a DNA sequence;
a nucleotide sequence wherein the DNA sequence comprises at least a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No. 8, preferably wherein the sequence is in isolated form;
a nucleotide sequence wherein the DNA sequence comprises at least a sequence that is the same as, or is complementary to, or has substantial homology with, or contains any suitable codon substitutions for any of those of, SEQ. ID. No. 12 or SEQ. ID. No. 13 or SEQ. ID. No. 14, preferably wherein the sequence is in isolated form; and
the use of beta-cyclodextrin to purify an enzyme, preferably GL.
Other preferred embodiments of the present invention include any one of the following: A transformed host organism having the capability of producing AF as a consequence of the introduction of a DNA sequence as herein described; such a transformed host organism which is a microorganismxe2x80x94preferably wherein the host
organism is selected from the group consisting of bacteria, moulds, fungi and yeast; preferably the host organism is selected from the group consisting of Saccharomyces, Kluyveromyces, Aspergillus, Trichoderma Hansenula, Pichia, Bacillus Streptomyces, Eschericia such as Aspergillus oryzae, Saccharomyces cerevisiae, bacillus sublilis, Bacillus amyloliquefascien, Eschericia coli.; A method for preparing the sugar 1,5-D-anhydrofructose comprising the use of a transformed host organism expressing a nucleotide sequence encoding the enzyme xcex1-1,4-glucan lyase, preferably wherein the nucleotide sequence is a DNA sequence, preferably wherein the DNA sequence is one of the sequences hereinbefore described; A vector incorporating a nucleotide sequence as hereinbefore described, preferably wherein the vector is a replication vector, preferably wherein the vector is an expression vector containing the nucleotide sequence downstream from a promoter sequence, preferably the vector includes a marker (such as a resistance marker); Cellular organisms, or cell line, transformed with such a vector; A method of producing the product xcex1-1,4-glucan lyase or any nucleotide sequence or part thereof coding for same, which comprises culturing such an organism (or cells from a cell line) transfected with such a vector and recovering the product.
In particular, in the expression systems, the enzyme should preferably be secreted to ease its purification. To do so the DNA encoding the mature enzyme is fused to a signal sequence, a promoter and a terminator from the chosen host.
For expression in Aspergillus niger the gpdA (from the Glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans) promoter and signal sequence is fused to the 5xe2x80x2 end of the DNA encoding the mature lyase. The terminator sequence from the A. niger trpC gene is placed 3xe2x80x2 to the gene (Punt, P. J. et al 1991xe2x80x94(1991): 3. Biotech. 17, 19-34). This construction is inserted into a vector containing a replication origin and selection origin for E. coli and a selection marker for A. niger. Examples of selection markers for A. niger are the amdS gene, the argB gene, the pyrG gene, the hygB gene, the BmlR gene which all have been used for selection of transformants. This plasmid can be transformed into A. niger and the mature lyase can be recovered from the culture medium of the transformants. Eventually the construction could be transformed into a protease deficient strain to reduce the proteolytic degradation of the lyase in the culture medium (Archer D. B. et al 992xe2x80x94Biotechnol. Lett. 14, 357-362).
Instead of Aspergillus niger as host, other industrial important microorganisms for which good expression systems are known could be used such as:Aspergillus oryzae, Aspergillus sp., Trichoderma sp., Saccharomyces cerevisiae, Kluyveromyces sp., Hansenula sp., Pichia sp., Bacillus subtilis, B. amyloliquefaciens, Bacillus sp., Streptomyces sp. or E. coli. 
The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB21 RY on Jun 20, 1994:
E. Coli containing plasmid pGL1 (NCIMB 40652)xe2x80x94[ref. DH5alpha-pGL1]; and
E. Coli containing plasmid pGL2 (NCIMB 40653)xe2x80x94[ref. DH5alpha-pGL2].
The following sample was accepted as a deposit in accordance with the Budapest Treaty at the recognised depositary The Culture Collection of Algae and Protozoa (CCAP) at Dunstaffnage Marine Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom, PA34 4AD on Oct. 11, 1994:
Fungally infected Gracilariopsis lemaneiformis (CCAP 1373/1)xe2x80x94[ref. GLQ-1 (Qingdao)].
Thus highly preferred embodiments of the present invention include a GL enzyme obtainable from the expression of the GL coding sequences present in plasmids that are the subject of either deposit NCIMB 40652 or deposit NCIMB 40653; and a GL enzyme obtainable from the fungally infected algae that is the subject of deposit CCAP 1373/1.
The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1 RY on Oct. 3, 1994:
E. Coli containing plasmid pMC (NCIMB 40687)xe2x80x94[ref. DH5alpha-pMC];
E. Coli containing plasmid pMV1 (NCIMB 40688)xe2x80x94[ref. DH5alpha-pMVI]; and.
E. Coli containing plasmid pMV2 (NCIMB 40689)xe2x80x94[ref. DH15alpha-pMV2].
Plasmid pMC is a pBluescript II KS containing a 4.1 kb fragment isolated from a genomic library constructed from Morchella costata. The fragment contains a gene coding for xcex1-1,4-glucan lyase.
Plasmid pMV1 is a pBluescript II KS containing a 2.45 kb fragment isolated from a genomic library constructed from Morchella vulgaris. The fragment contains the 5xe2x80x2 end of a gene coding for xcex1-1,4-glucan lyase.
Plasmid MV2 is a pPUC19 containing a 3.1 kb fragment isolated from a genomic library constructed from Morchella vulgaris. The fragment contains the 3xe2x80x2 end of a gene coding for xcex1-1,4-glucan lyase.
In the following discussions, MC represents Morchella costata and MV represents Morchella vulgaris. 
As mentioned, the GL coding sequence from Morchella vulgaris was contained in two plasmids. With reference to FIG. 15 pMV1 contains the nucleotides from position 454 to position 2902; and pMV2 contains the nucleotides downstream from (and including) position 2897. With reference to FIGS. 12 and 13, to ligate the coding sequences one can digest pMV2 with restriction enzymes EcoRI and BamHI and then insert the relevant fragment into pMV1 digested with restriction enzymes EcoRI and
Thus highly preferred embodiments of the present invention include a GL enzyme obtainable from the expression of the GL coding sequences present in plasmids that are the subject of either deposit NCIMB 40687 or deposit NCIMB 40688 and deposit NCIMB 40689.
The following sample was also accepted as a deposit in accordance with the Budapest Treaty at the recognised depositary The Culture Collection of Algae and Protozoa (CCAP) at Dunstaffnage Marine Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom, PA34 4AD on Oct. 11, 1994:
Fungally infected Gracilariopsis lemaneiformis (CCAP 1373/2)xe2x80x94[ref. GLSC-1 (California)].
Thus a highly preferred embodiment of the present invention includes a GL enzyme obtainable from the algae that is the subject of deposit CCAP 1373/2.
The present invention will now be described only by way of example.